Dec. 7, 2012
Finished the Run2 of the 306 ligand library. the top ten ligands are shown below
The best ranking list is shown from best to worst of fitness of the top ten ligands in the 306 library
Dec. 6, 2012
Enzyme assay using Suman's protein.
Did not work as well, the absorbance for 410nm was 0. Other people had similar results, so we think the protein had denature either due to
1.multiple times of defrosting
2.the glycerol mix was bad
3. it was not stored in glycerol at all
Suman's protein STSP was assayed twice. the first time it did not work because the values for the uM and molecular weight were incorrect, so the addition of tris base was incorrect. The second time, the correct values were entered, and that is when the above conclusions were made.
We have figured out how to fix the problem with defining the active site for GOLD, but i cannot do it on my Mac because I need a 3 click mouse editing. I will try to fix the problem tomorrow and try to screen the cb-kin library tomorrow if i can. but i did continue to do the second run on the 306 library, despite the active site problem.
Dec. 4, 2012
Enzyme assay with FTHAP, it was unsuccessful due to many reasons.
1. Bad technique
2. The protein was left in the water bath for too long
3. The protein is just not active
Absorbance shown of different concentrations. It should be linear, but because the absorbance was inconsistent at 410nm, we decided against continuing
the values are too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up, and we did not know which samples were which. Also there isn't a linear function as the concentrations of enzyme increased. I had also thrown out one of the samples because it was skewed from the rest of the data.
Dec. 3, 2012
Figured out virtual screening. The homology model i had made over the weekend was not a model of my protein, but a template. I screened the Cb-kin_UT library. Waiting for results.
Problems encountered:
an error message:
"init_protein: only found 43 solvent accessible atom in the active site: check origin and radius"
I am pretty sure this is what the error was referring to, so i am not sure how it docked or if it ever docked to my homology model. Although the rest of the output folders were fine. This was the only folder the list of "gold_soln" files.
112612 - ??. Dr B
Characterized the surrogate with Paul and Rishi Lane 1: Skipped Lane 2: 10-170kDa Protein Ladder Lane 3: Sample 1, Cell lysate after induction Lane 4: Sample 2, Soluble fraction Lane 5: Sample 3, Flow through Lane 6: Sample 4, Wash Lane 7: Elution 1 from Trial 1 (Tube A) Lane 8: Elution 2 from Trial 1 (Tube B) Lane 9-10: Skipped Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available.
Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1
Elution 1ubeB_RDvds.jpg
Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1
Elution 1tube A_RDvds.jpg
Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2
Elution 2tubeC_RDvds.jpg
Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2
Elution 2tubeD_RDvds.jpg
Week of Nov. 19, 2012
we did not want to spin down after sonicating until we were ready to characterize, and since there was thanksgiving break, we decided to wait until we all came back from break to continue. I have started on the homology model for virtual screening. I had a problem making it, because the program was not able to read the amino acid sequence i had entered.
Ling - latest results? - Dr. B 11/19/12 Week of Nov. 14, 2012
Expression was began on the evening of Nov. 14 where the bacteria cultures are grown up in the shaking incubator for 8 hours. The following day, Paul began on collecting data of the growth rate for the bacteria and induction. Me and Rishi centrifuged. Rishi had sonicated the solution.
Nov. 6, 2012 using the last of Paul's secondary to make pcr^2, the concentration of ___ ng/ul. But when we ran the gel, nothing had shown up. Also looking at the 260/280 ratio, it was not very close to 2, so there are probably contaminants.
Nov. 1, 2012
The concentrations were low and I used the template with the concentration of 144.9 ng/uL from Paul's PCR^2 clean up
PCR^2 after clean up tube A
PCR^2 after clean up tube B
Secondary PCR with Leishmania, used Paul's PCR^2 clean up for a template. A total of 8, 50 uL PCR tubes were made and used the original temperatures written in the protocol.
Paul's concentration of the PCR^2
Week 6
102112- Ling - ok show some results here.
-- Dr. B
Restart: Primary PCR and secondary PCR were done. I did do two secondary PCR at different annealing temperatures of 57C and 58C. I had lost my sample for 58C when I had stored it in the -20 fridge... So hopefully the 57C will come out on the agarose Gel
Well 1- skipped
Well 2-1Kb DNA ladder
Well 3- Primary PCR
Well 4- Secondary PCR
101612 - results ? -- Dr. B Week 5
100912 - Ling. Ok - work with Alex to see what you can figure out on making this work. You could try his Oligo mix, but I would recommend making a new fresh one. -- Dr. B
Secondary PCR 2nd time:
Oct. 2, 2012
The same results occurred the second time performing secondary PCR. I am thinking that i need to either change the temperature of the annealing proccess, or use someone else's oligo mix, because mine may have been made wrong.
Week 4
100112 - Ling - Results?? Dr .B
Secondary PCR on Sept 27, 2012
well 1- skipped, well 2- Secondary PCR
The secondary PCR did not actually show up at all. This has happened to one other person in the lab as well, the protocol will be repeated a second time, but the DNA ladder was really bright, so maybe there was too much Ethium Bromide, although i have consistently put 0.8 micro liters of EtBr.
Week 3
Ling - are you doing secondary PCR too. Dr. B
9/18/12 -
Well 1- Skipped, Well 2 - DNA ladder, Well 3 - Primary PCR
3rd time performing Primary PCR protocol. The smear should be longer, but it may still work, will comfirm if it works after secondary PCR.
Ling - include more description and legend labeling the gel and analysis of results. -- Dr. B 091812
Week 2
Sept 13, 2012
Primary PCR 1X TAE GEL:
The well 1- DNA ladder, well 2- primary PCR
Second time running primary PCR, 10mM of DNTP was used instead of 2mM. The primers did not anneal, which is why the band is so far down.
9/11/12 1st time PCR: the gel was not ran, because the oligo mix was diluted down to 0.01mM which is not enough to anneal.
Finished the Run2 of the 306 ligand library. the top ten ligands are shown below
Dec. 6, 2012
Enzyme assay using Suman's protein.
Did not work as well, the absorbance for 410nm was 0. Other people had similar results, so we think the protein had denature either due to
1.multiple times of defrosting
2.the glycerol mix was bad
3. it was not stored in glycerol at all
Suman's protein STSP was assayed twice. the first time it did not work because the values for the uM and molecular weight were incorrect, so the addition of tris base was incorrect. The second time, the correct values were entered, and that is when the above conclusions were made.
We have figured out how to fix the problem with defining the active site for GOLD, but i cannot do it on my Mac because I need a 3 click mouse editing. I will try to fix the problem tomorrow and try to screen the cb-kin library tomorrow if i can. but i did continue to do the second run on the 306 library, despite the active site problem.
Dec. 4, 2012
Enzyme assay with FTHAP, it was unsuccessful due to many reasons.
1. Bad technique
2. The protein was left in the water bath for too long
3. The protein is just not active
the values are too small to consider using for inhibition. During the collection of absorbance, the samples got mixed up, and we did not know which samples were which. Also there isn't a linear function as the concentrations of enzyme increased. I had also thrown out one of the samples because it was skewed from the rest of the data.
Dec. 3, 2012
Figured out virtual screening. The homology model i had made over the weekend was not a model of my protein, but a template. I screened the Cb-kin_UT library. Waiting for results.
Problems encountered:
an error message:
"init_protein: only found 43 solvent accessible atom in the active site: check origin and radius"
I am pretty sure this is what the error was referring to, so i am not sure how it docked or if it ever docked to my homology model. Although the rest of the output folders were fine. This was the only folder the list of "gold_soln" files.
112612 - ??. Dr B
Characterized the surrogate with Paul and Rishi
Lane 1: Skipped
Lane 2: 10-170kDa Protein Ladder
Lane 3: Sample 1, Cell lysate after induction
Lane 4: Sample 2, Soluble fraction
Lane 5: Sample 3, Flow through
Lane 6: Sample 4, Wash
Lane 7: Elution 1 from Trial 1 (Tube A)
Lane 8: Elution 2 from Trial 1 (Tube B)
Lane 9-10: Skipped
Some of Elution 2 spilled into Lane 9 causing faded bands. Also, Cell Lysate before induction was not inserted into Lane because not available.
Elution 1 (Concentration of 1.40 mg/mL): Tube A from Trial 1
Elution 2 (Concentration of 0.11 mg/mL): Tube B from Trial 1
Elution 1 (Concentration of 1.08 mg/mL): Tube C from Trial 2
Elution 2 (Concentration of 0.10 mg/mL): Tube D from Trial 2
Week of Nov. 19, 2012
we did not want to spin down after sonicating until we were ready to characterize, and since there was thanksgiving break, we decided to wait until we all came back from break to continue. I have started on the homology model for virtual screening. I had a problem making it, because the program was not able to read the amino acid sequence i had entered.
Ling - latest results? - Dr. B 11/19/12
Week of Nov. 14, 2012
Expression was began on the evening of Nov. 14 where the bacteria cultures are grown up in the shaking incubator for 8 hours. The following day, Paul began on collecting data of the growth rate for the bacteria and induction. Me and Rishi centrifuged. Rishi had sonicated the solution.
Nov. 6, 2012
using the last of Paul's secondary to make pcr^2, the concentration of ___ ng/ul. But when we ran the gel, nothing had shown up. Also looking at the 260/280 ratio, it was not very close to 2, so there are probably contaminants.
Nov. 1, 2012
The concentrations were low and I used the template with the concentration of 144.9 ng/uL from Paul's PCR^2 clean up
Secondary PCR with Leishmania, used Paul's PCR^2 clean up for a template. A total of 8, 50 uL PCR tubes were made and used the original temperatures written in the protocol.
Week 6
102112- Ling - ok show some results here.
-- Dr. B
Restart: Primary PCR and secondary PCR were done. I did do two secondary PCR at different annealing temperatures of 57C and 58C. I had lost my sample for 58C when I had stored it in the -20 fridge... So hopefully the 57C will come out on the agarose Gel
Well 1- skipped
Well 2-1Kb DNA ladder
Well 3- Primary PCR
Well 4- Secondary PCR
101612 - results ? -- Dr. B
Week 5
100912 - Ling. Ok - work with Alex to see what you can figure out on making this work. You could try his Oligo mix, but I would recommend making a new fresh one. -- Dr. B
Secondary PCR 2nd time:
Oct. 2, 2012
The same results occurred the second time performing secondary PCR. I am thinking that i need to either change the temperature of the annealing proccess, or use someone else's oligo mix, because mine may have been made wrong.
Week 4
100112 - Ling - Results?? Dr .B
Secondary PCR on Sept 27, 2012
The secondary PCR did not actually show up at all. This has happened to one other person in the lab as well, the protocol will be repeated a second time, but the DNA ladder was really bright, so maybe there was too much Ethium Bromide, although i have consistently put 0.8 micro liters of EtBr.
Week 3
Ling - are you doing secondary PCR too. Dr. B
9/18/12 -
3rd time performing Primary PCR protocol. The smear should be longer, but it may still work, will comfirm if it works after secondary PCR.
Ling - include more description and legend labeling the gel and analysis of results. -- Dr. B 091812
Week 2
Sept 13, 2012
Primary PCR 1X TAE GEL:
Second time running primary PCR, 10mM of DNTP was used instead of 2mM. The primers did not anneal, which is why the band is so far down.
9/11/12
1st time PCR: the gel was not ran, because the oligo mix was diluted down to 0.01mM which is not enough to anneal.